Colorimetric sensor arrays based on nanoporous pigments

ABSTRACT

A colorimetric array includes a substrate, a first spot on the substrate, and a second spot on the substrate. The first spot includes a first nanoporous pigment that includes a first nanoporous material and a first immobilized, chemoresponsive colorant. The second spot includes a second nanoporous pigment that includes a second nanoporous material and a second immobilized, chemoresponsive colorant. The first nanoporous pigment is different from the second nanoporous pigment.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.12/552,899, filed Sep. 2, 2009 and now issued as U.S. Pat. No. ______ on______, which claims benefit of U.S. Provisional Patent Application Ser.No. 61/094,301 entitled “Colorimetric Sensor Arrays Based on NanoporousPigments,” filed 4 Sep. 2008, the entire contents of both applicationswhich are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The subject matter of this application may in part have been funded bythe National Science Foundation (BES 05-28499) and Department of Defense(Army W91CRB-06-C-0018). The government may have certain rights in thisinvention.

BACKGROUND

Array based sensing has emerged as a powerful tool for the detection ofchemically diverse analytes. These systems mimic the mammalian gustatoryand olfactory systems by producing specificity, not from any singlesensor, but as a unique composite response for each analyte. Suchcross-reactive sensor arrays have application both as electronic nosetechnology for the detection of volatiles and gases [1-5], and aselectronic tongue technology for the detection of aqueous analytes[6-7].

Conventional sensor arrays typically have been based on a variety ofchanges in the properties of individual sensors. For example, absorptionof the analyte into conductive polymers or polymer composites can changethe electrical properties of the polymers or composites. In anotherexample, adsorption of the analyte onto surfaces such as metal oxidesurfaces can provide for combustion reactions, oxidation reactions orother electrochemical processes, which can be electrically detected. Inyet another example, a single fluorophore can be included in an array ofdifferent adsorbent polymers, and the change in composite fluorescenceof the array can be measured.

Using a different approach from these conventional sensor arrays,colorimetric sensor arrays are based on optoelectronics. A colorimetricsensor is a sensor that includes one or more materials that undergo achange in spectral properties upon exposure to an appropriate change inthe environment of the sensor. The change in spectral properties mayinclude a change in the absorbance, fluorescence and/or phosphorescenceof electromagnetic radiation, including ultraviolet, visible and/orinfrared radiation.

Colorimetric sensor arrays typically include an array of cross-reactivechemoresponsive dyes, where the colors of the chemoresponsive dyes areaffected by a wide range of analyte-dye interactions. Colorimetricarrays have been used for the identification and quantification of awide range of analytes, both in the gas phase and in aqueous solutions[8-17]. The arrays typically are made simply by printing the hydrophobicchemoresponsive dyes onto a hydrophobic membrane.

The chemoresponsive dyes used in colorimetric sensor arrays typicallyhave been limited to soluble molecular dyes, which are present in aporous film [18-23]. Insoluble, nonporous pigments have not providedsufficient contact between the analyte and the chromophores of thepigment, since the chromophores at the surface of the pigment are asmall fraction of the total number of chromophores. Likewise, nonporousfilms have not provided sufficient contact between a dye in the film andthe analyte in the sample.

There are a variety of drawbacks to the use of soluble molecular dyes inporous films for colorimetric sensor arrays. Aggressive solvents, suchas halocarbons or aromatics, are typically used for printing the dyes.The dyes can leach into analyte solutions from the porous film. The dyesmay be unstable, leading to a limited shelf-life. Crystallization of thedyes after printing on the membrane can render the dyes inactive.

One approach to addressing these drawbacks has been to immobilize thechemoresponsive dyes in sol-gel matrices [24-29]. These sol-gelmatrices, however, have had poor adherence to the hydrophobic surfacesused for sensor arrays. Thus, the sol-gel based dyes typically have beenprepared as a film or a monolithic disk, and have been used individuallyrather than as an array with other chemoresponsive dyes.

It would be desirable to provide a colorimetric sensor array havingincreased stability relative to conventional colorimetric arrays, andthat does not undergo leaching of soluble dyes during use. Ideally, sucha sensor array would include a variety of different chemoresponsivecolorants, including dyes and pigments. It would also be desirable forsuch an array to be formed by a method that does not include aggressivesolvents, and that is compatible with reproducible, high-throughputfabrication.

SUMMARY

In one aspect, the invention provides a calorimetric array including asubstrate, a first spot on the substrate, and a second spot on thesubstrate. The first spot includes a first nanoporous pigment thatincludes a first nanoporous material and a first immobilized,chemoresponsive colorant. The second spot includes a second nanoporouspigment that includes a second nanoporous material and a secondimmobilized, chemoresponsive colorant. The first nanoporous pigment isdifferent from the second nanoporous pigment.

In another aspect, the invention provides a method of making acolorimetric array including depositing a first liquid at a first pointon a substrate, depositing a second liquid at a second point on asubstrate, converting the first liquid into a first spot on thesubstrate, and converting the second liquid into a second spot on thesubstrate. The first spot includes a first nanoporous pigment includinga first nanoporous material and a first immobilized, chemoresponsivecolorant. The second spot includes a second nanoporous pigment includinga second nanoporous material and a second immobilized, chemoresponsivecolorant. The first nanoporous pigment is different from the secondnanoporous pigment.

These aspects may include a method of making a colorimetric array inwhich the first liquid includes a first nanoporous material precursorand a first chemoresponsive colorant, and the converting the firstliquid into a first spot includes solidifying the first nanoporousmaterial precursor to form the first nanoporous material. These aspectsmay include a method of making a colorimetric array in which the firstliquid includes a first colloidal suspension including a first solventand particles of the first nanoporous pigment, and the converting thefirst liquid into a first spot includes drying the first colloidalsuspension.

In yet another aspect, the invention provides a method of detecting ananalyte in a sample, including obtaining a first image of thecolorimetric array in the absence of the analyte, obtaining a secondimage of the colorimetric array in the presence of the sample, andanalyzing a difference between the first image and the second image.

The following definitions are included to provide a clear and consistentunderstanding of the specification and claims.

The term “colorant” means any material that absorbs light and/or thatemits light when exposed to higher frequency electromagnetic radiation.A light-absorbing portion of a colorant is referred to as a chromophore,and a light-emitting portion of a colorant is referred to as afluorophore.

The term “chemoresponsive colorant” means a colorant that undergoes achange in spectral properties in response to an appropriate change inits chemical environment.

The term “change in spectral properties” of a colorant means a change inthe frequency and/or intensity of the light the colorant absorbs and/oremits.

The term “dye” means a soluble colorant.

The term “pigment” means an insoluble colorant.

The term “nanoparticle” means a particle with one or more dimensions of100 nanometers (nm) or less.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood with reference to the followingdrawings and description. The components in the figures are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 is a schematic diagram of examples of two methods of making acolorimetric array.

FIG. 2 is a set of images of a slotted dip-pin printer for depositingliquids on a substrate.

FIG. 3A is an image from a colorimetric array, showing the array beforeexposure to a sample.

FIG. 3B is an image from a colorimetric array, showing the array afterexposure to a sample.

FIG. 3C is an image of a difference map of the two images from FIG. 3Aand

FIG. 3B.

FIG. 4 is a set of color difference maps from colorimetric arrays afterexposure to 16 different Toxic Industrial Chemicals (TICs) at their IDLH(immediately dangerous to life or health) concentrations.

FIG. 5 is a set of color difference maps from colorimetric arrays afterexposure to 3 different TICs at their IDLH concentrations, their PEL(permissible exposure level) concentrations, and well below their PELconcentrations.

FIG. 6 is a graph of the results of a Principal Component Analysis (PCA)of the quintuplicate tests of a colorimetric array against 16 differentTlCs at their IDLH concentration.

FIG. 7 illustrates a hierarchical cluster analysis of quintuplicatetests of a colorimetric array against 14 TICs, and a control sample.

FIG. 8 is a graph of the response time of a colorimetric array to sixdifferent representative sugars or sweeteners at 25 mM, as representedby the change in Euclidean distance over time.

FIG. 9 is a set of color difference maps from colorimetric arrays afterexposure to 14 sugars and sweeteners at 25 mM concentration, to sucroseat 150 mM concentration, and to a control.

FIG. 10 illustrates a hierarchical cluster analysis for quintuplicatetests of the colorimetric array against the 15 sugars and sweeteners ofFIG. 9, and a control.

FIG. 11 is a graph of the total Euclidean distance of the change inspectral properties of a colorimetric array as a function of D-glucoseconcentration.

FIG. 12 is a graph of the change in Euclidean distance over time, duringa repeated cycling of a colorimetric array between D-glucose (gray) anda buffer.

DETAILED DESCRIPTION

The present invention is based on the discovery that colorimetric arraysthat include nanoporous pigments can have increased stability relativeto conventional colorimetric arrays. Sensors including a colorimetricarray that includes nanoporous pigments also may have improvedsensitivity and selectivity toward analytes than conventionalcolorimetric arrays. In addition, the arrays including nanoporouspigments can provide a number of processing advantages.

A colorimetric array includes a substrate, a first spot on thesubstrate, and a second spot on the substrate. The first spot includes afirst nanoporous pigment including a first nanoporous material and afirst immobilized, chemoresponsive colorant. The second spot includes asecond nanoporous pigment including a second nanoporous material and asecond immobilized, chemoresponsive colorant. The second nanoporouspigment is different from the first nanoporous pigment.

The substrate may be any material that can retain a spot on its surface.Examples of substrates include polymeric membranes, such as celluloseacetate or polyvinylidene difluoride (PVDF). Examples of substratesinclude nonporous surfaces such as glass, metal, or a nonporous polymersurface such as poly(tetrafluoroethylene) (PTFE) or poly(ethyleneterephthalate) (PET).

The first nanoporous pigment includes a first nanoporous material and afirst immobilized, chemoresponsive colorant, and the second nanoporouspigment includes a second nanoporous material and a second immobilized,chemoresponsive colorant. The first and second nanoporous materials maybe the same, or they may be different. The first and second immobilized,chemoresponsive colorants also may be the same, or may be different. Inorder for the first and second nanoporous pigments to be different, atleast one of the nanoporous materials and the immobilized,chemoresponsive colorants must be different between the two spots. Ifthe first and second nanoporous materials are the same, then the firstand second immobilized, chemoresponsive colorants are different. If thefirst and second immobilized, chemoresponsive colorants are the same,then the first and second nanoporous materials are different. In oneexample, the first and second nanoporous materials are different, andthe first and second immobilized, chemoresponsive colorants also aredifferent.

The nanoporous material may be any material that includes pores,reticulations or void spaces with dimensions from 0.2 to 1000 nm.Preferably the nanoporous material includes pores with dimensions from0.5 to 100 nm. Preferably the nanoporous material includes pores thatare interconnected, such that a fluid can flow between the pores of thematerial. A nanoporous material may be, for example, an inorganicnetwork, such as a porous ceramic or a zeolite. A nanoporous materialmay be, for example, an organic network, such as a collection of carbontubes or a crosslinked gel. A nanoporous material may be, for example, amembrane material, such as a microfiltration membrane or anultrafiltration membrane. A nanoporous material may be a combination ofan inorganic network, an organic network and/or a membrane, such as aninorganic/organic composite.

The immobilized, chemoresponsive colorant may be any chemoresponsivecolorant that is immobilized as a part of a nanoporous pigment. Acolorant is immobilized as a part of a nanoporous pigment if less than1% of the colorant is extracted from the nanoporous pigment when incontact with a volume of water equal to or greater than the volume ofthe nanoporous pigment, for a period of 1 hour at room temperature.

The chemoresponsive colorant may be a chemoresponsive dye that isinsolubilized by the nanoporous material. Examples of chemoresponsivedyes include Lewis acid-base dyes, structure-sensitive porphyrins, pHsensitive dyes, solvatochromic dyes, vapochromic dyes, redox sensitivedyes, and metal ion sensitive dyes. Chemoresponsive dyes may beresponsive to one or more chemical interactions including Lewisacid-base interaction, Brønsted acid-base interaction, ligand binding,π-π complexation, hydrogen bonding, polarization, oxidation/reduction,and metal coordination.

The chemoresponsive dye may be insolubilized by the nanoporous materialthrough a chemical bond, such as a covalent bond, an ionic bond or ahydrogen bond. The chemoresponsive dye may be insolubilized by thenanoporous material through adsorption on a surface of the nanoporousmaterial. The chemoresponsive dye may be insolubilized by the nanoporousmaterial through physical entrapment of the dye in the nanoporousmaterial. The chemoresponsive dye may be on an exterior surface of thenanoporous material, or it may be on an interior surface, such as withinthe pores, reticulations or void spaces of the material.

The chemoresponsive dye may be, for example, a Lewis acid-base dye, suchas a Lewis acid dye or a Lewis base dye. A Lewis acid-base dye is a dyethat can interact with a substance by acceptor-donor sharing of a pairof electrons with the substance, resulting in a change in spectralproperties. The change in spectral properties for a Lewis acid-base dyemay be related to Lewis acid-base interaction and ligand binding, butalso to π-π complexation, hydrogen bonding, and/or polarity changes.Lewis acid-base dyes include metal-ion containing dyes, such asmetalloporphyrins and other metal ion ligating macrocycles or chelatingdyes; boron- and boronic acid containing dyes; and dyes with accessibleheteroatoms (e.g., N, O, S, P) with lone electron pairs capable of Lewiscoordination (e.g., “complexometric dyes”).

Examples of Lewis acid-base dyes include metal ion-containing dyes, suchas metal ion-containing porphyrins (i.e., metalloporphyrins), salencomplexes, chlorins, bispocket porphyrins, and phthalocyanines.Diversity within the metalloporphyrins can be obtained by variation ofthe parent porphyrin, the porphyrin metal center, or the peripheralporphyrin substituents. The parent porphyrin is also referred to as afree base porphyrin, which has two central nitrogen atoms protonated(i.e., hydrogen cations bonded to two of the central pyrrole nitrogenatoms). In one example, a parent porphyrin is the so-called free baseform 5,10,15,20-tetraphenylporphyrin (H2TPP), its dianion is5,10,15,20-tetraphenyl-porphyrinate(-2) (TPP dianion), its metalatedcomplexes, and its acid forms (H₃TPP+ and H₄TPP⁺²). This porphyrin mayform metalated complexes, for example, with Sn⁴+, Co³+, Co²+, Cr+³,Mn+³, Fe+³, Cu²+, Ru²+, Zn²+, Ag²+, In³+, and Ir+³. Metal ion-containingmetalloporphyrin dyes are described, for example, in U.S. Pat. No.6,368,558 81 to Suslick et al. and in U.S. Patent ApplicationPublication No. 2003/0143112 A1 to Suslick et al.

Visible spectral shifts and absorption intensity differences formetalloporphyrins may occur upon ligation of the metal center, leadingto readily observable changes in spectral properties. The magnitude ofthis spectral shift typically correlates with the polarizability of theligand, thus allowing for distinction between analytes based on theelectronic properties of the analytes. Using metal centers that span arange of chemical hardness and ligand binding affinity, it may bepossible to differentiate between a wide range of volatile analytes,including molecules having soft functional groups such as thiols, andmolecules having hard functional groups such as amines. Becauseporphyrins can exhibit wavelength and intensity changes in theirabsorption bands with varying solvent polarity, an array that includesporphyrins may be used to colorimetrically distinguish among a series ofweakly ligating solvent vapors, such as arenes, halocarbons and ketones.

The chemoresponsive dye may be, for example, a structure-sensitiveporphyrin. Structure-sensitive porphyrins include modified porphyrinsthat include a super structure bonded to the periphery of the porphyrin.For example, metalloporphyrins functionalized with a super structure atthe periphery may limit steric access to the metal ion, allowing forshape-selective distinction of analytes, such as between n-hexylamineand cyclohexylamine. Controlling the ligation of various nitrogenousligands to dendrimer-metalloporphyrins can provide for selectivitiesover a range of more than 10⁴.

Examples of super structures that may be bonded to a porphyrin includedendrimers, siloxyl groups, aryl groups such as phenyl groups, alkylgroups such as t-butyl groups, organometallic groups, inorganic groups,and other bulky substituents. Porphyrins bearing super structures may beselective to molecular shape, including sensitivity to steric factors,enantiomeric factors, and regioisomeric factors. For example, thestructures may provide stericallly constrained pockets on one or bothfaces of the porphyrin. Porphyrins bearing super structures also may besensitive to factors such as hydrogen bonding and acid-basefunctionalities. Metal ion-containing metalloporphyrin dyes that includea super structure bonded to the periphery of the porphyrin, and methodsof making such dyes, are disclosed, for example, in U.S. Pat. No.6,495,102 B1 to Suslick et al.

One example of modified porphyrins that include a super structure bondedto the periphery of the porphyrins is the family oftetrakis(2,4,6-trimethoxyphenyl)-porphyrin (TTMPP). By varying the metalin this porphyrin, it is possible to distinguish between substances suchas between t-butylamine and n-butylamine, and between cyclohexylamineand n-hexylamine.

Another example of a modified porphyrin that includes a super structurebonded to the periphery of the porphyrin is the family ofsilylether-metalloporphyrins. For example, scaffolds derived from thereaction of 5,10,15,20-tetrakis(2′,6′-dihydroxyphenyl)-porphyrinatozinc(II) with t-butyldimethylsilyl chloride provide Zn (II) porphyrin havingin which the two faces are protected with six, seven, or eight siloxylgroups. This can result in a set of three porphyrins having similarelectronic properties, but having different hindrance around the centralmetal atom present in the porphyrin. The shape selectivities of theseporphyrins may be up to 10⁷ or greater.

Other examples of a modified porphyrins that include a super structurebonded to the periphery of the porphyrin include siloxyl-substitutedbis-pocket porphyrins, such as5-phenyl-10,15,20-tris(2′,6′-dihydroxyphenyl)porphyrinatozinc(II);5,10,15,20-tetrakis(2′,6′-dihydroxyphenyl)porphyrinatozinc(II);5(phenyl)-10,15,20-trikis(2′,6′-disilyloxyphenyl)porphyrinatozinc(II);5,10,15-trikis(2′,6′-disilyloxyphenyl)-20-(2′-hydroxy-6′-silyloxyphenyl)porphyrinatozinc(II).The shape selectivities of these porphyrins may be up to 107 or greatercompared to unhindered metalloporphyrins. Fine-tuning of ligationproperties of these porphyrins may be possible, such as by using pocketsof varying steric demands.

Other examples of metal ion-containing metalloporphyrin dyes thatinclude a super structure bonded to the periphery of the porphyrininclude2,3,7,8,12,13,17,18-octafluoro-5,10,15,20-tetrakis(pentafluorophenyl)-porphyrinatocobalt(II);2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetraphenyl-porphyrinatozinc(II); 5,10,15,20-tetraphenylporphyrinatozinc (II);5(phenyl)-10,15,20-trikis(2′,6′-bis(dimethyl-t-butylsiloxyl)phenyl)porphyrinatozinc(II);5,110,15,20-tetrakis(2′,6′-bis(dimethyl-t-butylsiloxyl)phenyl)porphyrinatozinc(II); 5,10,15,20-tetraphenylporphyrinatocobalt (II);5,10,15,20-tetrakis(2,6-difluorophenyl)-porphyrinatozinc(II); and5,10,15,20-tetrakis(2,4,6-trimethylphenyl)-porphyrinatozinc (II).

An array that includes a structure-sensitive porphyrin may be used incombinatorial libraries for shape selective detection of substrates.Such an array also may include a structure-sensitive having chiral superstructures on the periphery of the porphyrin, which may provide foridentification of chiral substrates, such as drugs, natural products andcomponents of biological samples from a patient. Such an array also maybe used for analysis of biological entities based on the surfaceproteins, oligosaccharides, antigens, etc., that interact with themetalloporphyrins. Examples of biological entities include individualspecies of bacteria and viruses. Such an array also may be used foranalysis of nucleic acid sequences, including specific recognition ofindividual sequences of nucleic acids. Substituents on the porphyrinsthat would be particularly useful in this regard include known DNAintercalating molecules and nucleic acid oligomers.

The chemoresponsive dye may be, for example, a pH sensitive dye. Dyesthat are pH sensitive include pH indicator or acid-base indicator dyesthat may change color upon exposure to acids or bases. Examples of pHsensitive dyes include Brønsted acid dyes. A Brønsted acid dye is aproton donor that can donate a proton to a Brønsted base (i.e., a protonacceptor), resulting in a change in spectral properties. Under certainpH conditions, a Brønsted acid dye may be a Brønsted base.

Examples of Brønsted acid dyes include protonated, but non-metalated,porphyrins; chlorines; bispocket porphyrins; phthalocyanines; andrelated polypyrrolic dyes. Examples of non-metalated porphyrin Brønstedacid dyes include5,10,15,20-tetrakis(2′,6′-bis(dimethyl-t-butylsiloxyl)phenyl)porphyrindication; 5,10,15,20-Tetraphenyl-21H,23H-porphyrin; or5,10,15,20-Tetraphenylporphyrin dication. Other examples of Brønstedacid dyes include Chlorophenol Red, Bromocresol Green, BromocresolPurple, Bromothymol Blue, Bromopyrogallol Red, Pyrocatechol Violet,Phenol Red, Thymol Blue, Cresol Red, Alizarin, Mordant Orange, MethylOrange, Methyl Red, Congo Red, Victoria Blue B, Eosin Blue, Fat Brown B,Benzopurpurin 4B, Phloxine B, Orange G, Metanil Yellow, Naphthol GreenB, Methylene Blue, Safranine O, Methylene Violet 3RAX, Sudan Orange G,Morin Hydrate, Neutral Red, Disperse Orange #25, Rosalie Acid, Fat BrownRR, Cyanidin chloride, 3,6-Acridineamine,6′-Butoxy-2,6-diamino-3,3′-azodipyridine, para-Rosaniline Base, AcridineOrange Base, Crystal Violet, Malachite Green Carbinol Base, Nile Red,Nile Blue, Nitrazine Yellow, Bromophenol Red, Bromophenol Blue,Bromoxylenol Blue, Xylenol Orange Tetrasodium Salt,1-[4-[[4-(dimethylamino)phenyl)azo]phenyl)-2,2,2-trifluoro-ethanone,4-[2-[4-(dimethylamino)phenyl]ethenyl)-2,6-dimethyl-pyryliumperchlorate, and 1-amino-4-(4-decylphenylazo)-naphthalene.

The chemoresponsive dye may be, for example, a solvatochromic dye or avapochromic dye. Solvatochromic dyes may change color depending upon thelocal polarity of their liquid micro-environment. Vapochromic dyes maychange color depending upon the local polarity of their gaseousmicro-environment. Most dyes are solvatochromic and/or vapochromic tosome extent; however, some are much more responsive than others,especially those that can have strong dipole-dipole interactions.Examples of solvatochromic dyes include Reichardt's Dyes, Nile Red,Fluorescein, and polypyrrolic dyes.

An array that includes a pH sensitive dye and/or a solvatochromic orvapochromic dye may be useful in differentiating analytes that do notbind to, or bind only weakly to, metal ions. Such analytes includeacidic compounds, such as carboxylic acids, and certain organiccompounds lacking ligatable functionality. Examples of organic compoundslacking ligatable functionality include simple alkanes, arenes, and somealkenes and alkynes, especially if sterically hindered. Examples oforganic compounds lacking ligatable functionality also include moleculesthat are sufficiently sterically hindered to preclude effectiveligation. Arrays that include a pH sensitive and/or a solvatochromic orvapochromic dye are described, for example, in U.S. Patent ApplicationPublication No. 2003/0143112 A1 to Suslick et al.

The chemoresponsive dye may be, for example, a redox sensitive dye thatundergoes a change in spectral properties depending upon its oxidationstate. Examples of dyes that are redox sensitive include redoxindicators are disclosed in H. A. Laitinen, Chemical Analysis(McGraw-Hill: New York, 1960). Examples of redox indicators includemethylene blue, naphthol blue-black, brilliant ponceau,α-naphthoflavone, basic fuchsin, quinoline yellow, thionin acetate,methyl orange, neutral red, diphenylamine, diphenylaminesulfonic acid,1,10-phenanthroline iron(II), permanganate salts, silver salts, andmercuric salts.

The chemoresponsive dye may be, for example, a metal ion sensitive dyethat undergoes a change in spectral properties in the presence of metalions. Examples of dyes that are metal ion sensitive include metal ionindicators are disclosed in Laitinen [30]. Examples of metal ionindicator dyes include eriochrome black T, murexide,1-(2-pyridylazo)-2naphthol, and pyrocatechol violet.

The chemoresponsive colorant may be a chemoresponsive pigment.Preferably the chemoresponsive pigment is a porous pigment. A porouspigment particle has a chemoresponsive surface area that is much greaterthan the chemoresponsive surface area of a corresponding nonporouspigment particle. Examples of porous pigments include porous calciumcarbonate, porous magnesium carbonate, porous silica, porous alumina,porous titania, and zeolites.

The chemoresponsive colorant may be a chemoresponsive nanoparticle. Achemoresponsive nanoparticle may be a discrete nanoparticle, or it maybe formed from nanoparticle-forming ions or molecules. Examples ofnanoparticle-forming ions or molecules are disclosed in Murphy et al.[31]. The nanoparticle may be in a variety of forms, including ananosphere, a nanorod, a nanofiber, and a nanotube. Examples ofchemoresponsive nanoparticles include nanoporous porphyrin solids,semiconductor nanoparticles such as quantum dots, and metalnanoparticles. Examples of nanoporous porphyrin solids are disclosed inSuslick et al. [32].

A colorimetric array may further include a plurality of additional spotson the substrate, where each additional spot independently includes achemoresponsive colorant. At least one spot of the additional spots mayinclude an additional nanoporous pigment that is different from thefirst and second nanoporous pigments. An additional nanoporous pigmentincludes a nanoporous material and an immobilized, chemoresponsivecolorant. The nanoporous material and the colorant may be as describedabove for nanoporous materials and chemoresponsive colorants.Preferably, each additional spot independently includes an additionalnanoporous pigment that is different from the first and secondnanoporous pigments. More preferably, each additional spot independentlyincludes an additional nanoporous pigment, each of the additionalnanoporous pigments is different, and each of the additional nanoporouspigments is different from the first and second nanoporous pigments. Asnoted above, two nanoporous pigments are different if their componentnanoporous materials and/or their immobilized, chemoresponsive colorantsare different.

A colorimetric array that further includes a plurality of additionalspots on the substrate may include at least one spot that does notinclude a nanoporous pigment. For example, the array may include atleast one spot including a chemoresponsive colorant that is notimmobilized with a nanoporous material. Colorimetric arrays in which thespots do not include nanoporous pigments are disclosed, for example inU.S. Pat. Nos. 6,368,558 and 6,495,102 to Suslick et al., and in U.S.Patent Application Publication Nos. 2003/0143112, 2003/0129085 and2003/0166298 to Suslick et al. [18-23]. Thus, a single colorimetricarray may include spots including nanoporous pigments, and also mayinclude spots that include only chemoresponsive colorants that are notimmobilized with a nanoporous material.

The use of more than one type of chemoresponsive colorant may expand therange of analytes to which the array is sensitive, may improvesensitivity to some analytes, and/or may increase the ability todiscriminate between analytes. In one example, a colorimetric arrayincludes from 2 to 1,000 spots. Preferably a colorimetric array includesfrom 4 to 500 spots. More preferably, a colorimetric array includes from8 to 250 spots. More preferably, a colorimetric array includes from 10to 100 spots. More preferably, a colorimetric array includes from 16 to49 spots, including from 36 spots. Each spot in a colorimetric array mayinclude a different colorant. However, it may be desirable to includeduplicate spots that include the same colorant. Duplicate spots may beuseful, for example, to provide redundancy to the array and/or to serveas an indicator for quality control.

A method of making a colorimetric array includes depositing a firstliquid at a first point on a substrate, depositing a second liquid at asecond point on a substrate, converting the first liquid into a firstspot on the substrate, and converting the second liquid into a secondspot on the substrate. The first spot includes a first nanoporouspigment including a first nanoporous material and a first immobilized,chemoresponsive colorant. The second spot includes a second nanoporouspigment including a second nanoporous material and a second immobilized,chemoresponsive colorant. The second nanoporous pigment is differentfrom the first nanoporous pigment.

In a first example, the first liquid includes a first nanoporousmaterial precursor and a first chemoresponsive colorant. A nanoporousmaterial precursor is a substance that will form a nanoporous materialwhen it is solidified. In this example, converting the first liquid intoa first spot includes solidifying the first nanoporous materialprecursor to form the first nanoporous material. The first nanoporousmaterial precursor may be, for example, a polymer, a prepolymer, ceramicprecursors, or mixtures of these. The first liquid may include otheringredients, such as a solvent and/or a surfactant.

The first nanoporous material precursor may include starting materialsfor a ceramic that have been at least partially hydrolyzed. The firstliquid may be formed by combining ingredients including startingmaterials for a ceramic, a solvent, and the first chemoresponsivecolorant to form a first mixture, and then hydrolyzing the first mixtureto form a sol. Solidifying the first nanoporous material precursor mayinclude condensing the first nanoporous material precursor to form agel, and drying the gel to form the first nanoporous material.

The solidifying may include any method that converts the nanoporousmaterial precursor into a nanoporous material. Examples ofsolidification methods include chemical crosslinking, exposure to UVradiation, and heating. In one example, the solidifying includes heatingthe liquid on the substrate. Initial curing at room temperature for 24to 72 hours may be preferred in order to maintain porosity of thenanoporous pigment. Additional heating may be performed, for example, ina standard convection oven. If the substrate is temperatur,e-sensitive,heating the liquid for 24 hours at temperatures lower than 70° C. ispreferred. When preparing a spot that includes a nanoporous ceramic anda pH responsive dye as the chemoresponsive colorant, solidifying at 60°C. or even at room temperature is preferred. With more thermally robustsubstrates, solidifying may be completed much more rapidly, for examplein 1 hour at 120° C.

The second liquid may be as described for the first liquid, and may beconverted into the second spot in a similar way. For example, the secondliquid may include a second nanoporous material precursor and a secondchemoresponsive colorant, and converting the second liquid into a secondspot may include solidifying the second nanoporous material precursor toform the second nanoporous material. The second nanoporous materialprecursor may be as described for the first nanoporous materialprecursor. Alternatively, the second liquid may include a colloidalsuspension that is converted into a second spot by drying, as describedbelow.

FIG. 1 is a schematic diagram, the top pathway of which is an example ofthis method. In this pathway, an initial liquid is formed by combiningsilica precursors, a chemoresponsive colorant, and a solvent includingwater. The liquid undergoes hydrolysis to form a colloidal sol, and isthen deposited at a point on a substrate. The silica precursors aresolidified by condensation to form a silica gel, followed by drying thegel to form a silica aerogel. The spot formed is thus the nanoporousaerogel including the immobilized, chemoresponsive colorant.

In a second example, the first liquid includes a first colloidalsuspension including a first solvent and particles of the firstnanoporous pigment. In this example, converting the first liquid into afirst spot includes drying the first colloidal suspension. The firstliquid may be formed by dispersing the particles of the first nanoporouspigment in the first solvent. The first liquid may include otheringredients, such as a surfactant.

The particles of the first nanoporous pigment may be formed by combiningingredients including starting materials for a ceramic, a secondsolvent, and the first chemoresponsive colorant to form a first mixture;hydrolyzing the first mixture to form a sol; and condensing the sol toform a second mixture including the particles of the first nanoporouspigment.

The second liquid may be as described for the first liquid, and may beconverted into the second spot in a similar way. For example, the secondliquid may include a second colloidal suspension including a thirdsolvent and particles of the second nanoporous pigment, and convertingthe second liquid into a second spot may include drying the secondcolloidal suspension. The second liquid may be formed by dispersing theparticles of the second nanoporous pigment in the third solvent. Theparticles of the second nanoporous pigment may be as described for theparticles of the first nanoporous pigment.

Referring to FIG. 1, the bottom pathway is an example of this method. Inthis pathway, an initial liquid is formed by combining silicaprecursors, a chemoresponsive colorant, and a solvent including water.The preliminary liquid undergoes hydrolysis to form a colloidal sol, andthen undergoes condensation and precipitation to form nanoporous pigmentparticles that include the chemoresponsive colorant in an immobilizedstate. A liquid is then formed by dispersing the nanoporous pigmentparticles and a surfactant in a solvent. This liquid is deposited at apoint on a substrate, and then dried to form a silica aerogel. The spotformed is thus the aerogel including the chemoresponsive colorant.

In the method of forming a colorimetric array, the first and secondliquids, and mixtures used to form the liquids, independently mayinclude a solvent. Examples of solvents include 1,2-dichlorobenze,diglyme, methanol, 2-methoxyethanol, propylene glycol methyl etheracetate, water, and mixtures of these. Preferably the liquids include asolvent that contains a mixture of water and another solvent.

In the method of forming a colorimetric array, the first and secondliquids, and mixtures used to form the liquids, independently mayinclude a surfactant. A surfactant may be useful to enhance thesolubility or dispersibility of the chemoresponsive colorant in aliquid, to improve flow control, to enhance the uniformity of theprinting or of the color (i.e., “leveling”), and/or to improve wettingof the surface being printed (i.e., “wet-out”). The surfactant may becationic, anionic, zwitterionic, or nonionic. Examples of surfactantsinclude sodium dodecyl sulfate (SDS) and Triton X-100, a GE™ Silwet™silicone surfactant, a 3M™ Novec™ fluorosurfactant, and mixtures ofthese.

Examples of starting materials for a ceramic include starting materialsfor materials such as hydroxyapatite, titanium oxide, lead zirconate,titanate, alumina, silica, zirconia, silicon nitride, barium titanate,and silicon carbide, or mixtures of these. In one example, startingmaterials for a silica ceramic may include at least one alkoxysilanes orhalosilanes, and including a condensation catalyst. The alkoxysilane orhalosilane may be, for example, tetramethoxysilane (TMOS),tetrachlorosilane (TCS), methyltrimethoxysilane (MTMS),methyltrichlorosilane (MTCS), octyltrimethoxysilane (OTES),phenethyltrimethoxysilane (PTMS), or mixtures of these. The condensationcatalyst may be, for example, an acid such as hydrochloric acid (HCl) ornitric acid (HNO₃), or a base such as ammonium hydroxide (NH4OH) orsodium hydroxide (NaOH), dissolved in water.

In a specific example, an initial mixture includes 5 to 50% by volumealkoxysilanes, 0.001 to 0.1 M hydrochloric acid as a condensationcatalyst, a chemoresponsive colorant, 5 to 20% by volume water, 1 to 80%by volume solvents, and 0.01 to 2% by weight surfactant. This initialmixture may be hydrolyzed to form a sol, deposited on a substrate,condensed to form a gel, and dried to form a spot of an array.Alternatively, this initial mixture may be hydrolyzed to form a sol, andcondensed to form a mixture that includes particles of a nanoporouspigment. These particles may then be dispersed in a solvent, depositedon a substrate, and dried to form a spot of an array.

The depositing may include one or more printing techniques such asink-jet, stamping and dip-pin printing. FIG. 2 is a set of images of aslotted dip-pin printer for depositing liquids on a substrate. Each ofthe liquids may be deposited on a single substrate. Alternatively,different liquids may be printed on different substrates, and theindividual substrates then assembled together to form the array.

A method of making a colorimetric array may further include depositing aplurality of additional liquids at additional points on the substrate,and converting the additional liquids into a plurality of additionalspots. Each additional spot independently includes a chemoresponsivecolorant. At least one of the additional spots may include an additionalnanoporous pigment that is different from the first and secondnanoporous pigments. An additional nanoporous pigment includes ananoporous material and an immobilized, chemoresponsive colorant. Thenanoporous material and the colorant may be as described above fornanoporous materials and chemoresponsive colorants. Preferably, eachadditional spot independently includes an additional nanoporous pigmentthat is different from the first and second nanoporous pigments. Morepreferably, each additional spot independently includes an additionalnanoporous pigment, each of the additional nanoporous pigments isdifferent, and each of the additional nanoporous pigments is differentfrom the first and second nanoporous pigments. As noted above, twonanoporous pigments are different if their component nanoporousmaterials and/or their immobilized, chemoresponsive colorants aredifferent.

The method may include depositing from 2 to 1,000 liquids. Preferablythe method includes depositing from 4 to 500 liquids. More preferably,the method includes depositing from 8 to 250 liquids. More preferably,the method includes depositing from 10 to 100 liquids. More preferably,the method includes depositing from 16 to 72 liquids, including from 24to 36 liquids. Each liquid may include a different colorant. However, itmay be desirable to include duplicate liquids that include the samecolorant, as noted above.

A colorimetric array that includes first and second spots, eachincluding a different nanoporous pigment, may be used for chemicalanalyses of gaseous and liquid analytes. A method of detecting ananalyte in a sample includes obtaining a first image of the array in theabsence of the analyte, obtaining a second image of the array in thepresence of the sample, and analyzing a difference between the firstimage and the second image. The array may be used to detect a widevariety of analytes, regardless of the physical form of the analytes.The array may be used to detect any vapor emitting substance, includingliquid, solid, or gaseous substances, and even when mixed with othervapor emitting substances. The array may be used to detect analytesdissolved in a solvent, including analytes in water. The array may beused to detect ionic or molecular analytes in a solvent, even when mixedwith other dissolved analytes.

Obtaining an image of a colorimetric assay can be performed with anysuitable imaging device. Examples of imaging devices include flatbedscanners, digital cameras (preferably with either constant illuminationor reproducible flash intensity), CCD or CMOS video cameras (alsopreferably with reproducible illumination such as LED, white ortricolor). A handheld device can be constructed to read the internalsensor array with internal computing capability (for example a pocket PCor an embedded microprocessor), a light source and an imaging camera.

For gas or vapor analytes, a gas stream containing the analyte is passedover the array, and images may be obtained before, during and/or afterexposure to the gas stream. Preferably an image is obtained after thesample and the array have equilibrated. If the gas stream is notpressurized, it may be useful to use a miniaturized pump.

For analytes dissolved in a solvent, either aqueous or non-aqueous, thefirst image may be obtained in air or, preferably, after exposure to thepure carrier solvent that is used of the sample. The second image of thearray may be obtained after the start of the exposure of the array tothe sample. Preferably an image is obtained after the sample and thearray have equilibrated.

Analyzing the differences between the first image and the second imagemay include quantitative comparison of the digital images before andafter exposure to the analyte. Using customized software such asChemEye™ (ChemSensing, Champaign, Ill.) or standard graphics softwaresuch as Adobe® PhotoShop®, a difference map can be obtained bysubtracting the first image from the second image. To avoid subtractionartifacts at the periphery of the spots, the center of each spot can beaveraged.

FIGS. 3A-3C are images from a colorimetric array, showing the arraybefore exposure to a sample (3A), after exposure to a sample (3B), and adifference map of these two images (3C). The comparison data obtainedfrom the difference map includes changes in red, green and blue values(f1RGB) for each spot in the array. Preferably the changes in spectralproperties that occur upon exposure to an analyte, and the resultantcolor difference map, can serve as a unique fingerprint for any analyteor mixture of analytes at a given concentration.

In the simplest case, an analyte can be represented by a single 3×vector representing the ΔRGB values for each colorant, where xis thenumber of colorants as set forth in equation (1). This assumes thatequilibration is relatively rapid and that any irreversible reactionsbetween analyte and colorant are slow relative to the initialequilibration time

Difference vector=ΔR1, ΔG1, ΔB1, ΔR2, ΔG2, ΔB2, ΔRx, ΔGx, ΔBx  (1).

Alternatively, the temporal response of the analyte can be used to makerapid identification, preferably using a “time-stack vector” of ΔRGBvalues as a function of time. In equation 2, a time-stack vector isshown for an array of 36 colorants at times m, n, and finally z, allusing the initial scan as the baseline for the differences in red, greenand blue values:

Time stack vector=ΔR1m, ΔG1m, ΔB1m, ΔR2m, ΔG2m, ΔB2m, ΔR36m, ΔG36m,ΔB36m, . . . ΔR1n, ΔG1n, ΔB1n, . . . , ΔR36m, ΔG36m, ΔB36m, . . . ,ΔR36z, ΔG36z, ΔB36z  (2).

Accordingly, each analyte response can be represented digitally as avector of dimension 3xz, where x is the number of colorants and z is thenumber of scans at different times. Quantitative comparison of suchdifference vectors can be made simply by measuring the Euclideandistance in the 3xz space. Such vectors may then be treated by usingroutine chemometric or statistical analyses, including principalcomponent analysis (PCA), hierarchical cluster analysis (HCA) and lineardiscriminant analysis. Statistical methods suitable for highdimensionality data are preferred. As an example, HCA systematicallyexamines the distance between the vectors that represent each colorant,forming clusters on the basis of the multivariate distances between theanalyte responses in the multidimensional ΔRGB color space using theminimum variance (“Ward's”) method for classification [33]. A dendrogramcan then be generated that shows the clustering of the data from theEuclidean distances between and among the analyte vectors, much like anancestral tree.

A method of detecting an analyte may include forming a derivative of theanalyte of interest. This may be useful when the original analytes proverelatively non-responsive to the array colorants. In this case, achemical reaction of the analyte may form one or more products that arewell detected by the array colorants.

In one example, the response of arrays to various sugars dissolved inwater may not be sufficient for direct analysis. However, the reactionof sugar analytes with boronic or boric acids yields secondary analytesthat can be analyzed on an array. Without being bound to any particulartheory, it appears that this may be ascribed to the fact that differentcarbohydrates have different association constants to boronic acid,leading to changes in solution pH [34-37].

In another example, an analyte may be partially oxidized prior toobtaining the second image of the colorimetric array. Partial oxidationof an analyte means oxidation that does not convert all of the carbonatoms of the analytes completely to carbon dioxide. By partiallyoxidizing analytes, new mixtures of partially oxidized analytes areformed that may provide a unique analytical fingerprint for the presenceof the parent analytes. Partial oxidation may include contacting theanalyte with an oxidizing agent, such as oxygen gas, hydrogen peroxide,hypochlorite, chlorine dioxide, chlorine, and optionally may includecontacting the analyte with an oxidation catalyst. Preferably theoxidizing source is present at a concentration or amount that issufficient to result in forming an oxidized analyte, but that is belowthat needed to fully oxidize the parent analyte completely to carbondioxide. Colorimetric analysis using an array, where the analyte ispartially oxidized, is described, for example, in U.S. PatentApplication Publication No. 2003/0166298 A1 to Suslick et al.

Partial oxidation may form a mixture of alcohols, aldehydes, ketones,carboxylic acids, carbon monoxide, and/or carbon dioxide. For example, asample including a weakly-responsive analyte can be converted to atleast one partially oxidized analyte that is more volatile. In oneexample, hexane can be partially oxidized to derivative analytes such ashexanoic acid, hexanol, hexanal, and C5-ketones. The more volatileorganic compounds typically have a stronger interaction with the array,and thus may provide stronger responses than the parent analytes.

A colorimetric array that includes first and second spots, eachincluding a nanoporous material and a chemoresponsive colorant, may beused to detect ammonia or other analytes in exhaled breath. Detection ofammonia in exhaled breath can be useful in detecting the presence of aHelicobacter infection or for diagnosing liver or renal function. Thecolorimetric detection of ammonia in exhaled breath is described, forexample, in U.S. Patent Application Publication No. 2005/0171449 A1,with inventors Suslick et al.

A colorimetric array may be placed in a clear plastic cartridge, and thecartridge may then be sealed to protect the array from the ambientenvironment. A gaseous or liquid sample can be introduced by injectingthe sample into the cartridge. Alternatively, a colorimetric array maybe formed on a substrate, and a cover may be attached to the substrateto form a sealed chamber that encloses the array and that includes aninlet port, and optionally an exit port. Cartridges for colorimetricarrays are disclosed in co-pending U.S. Provisional Patent ApplicationNo. 61/094,311, filed Sep. 4, 2008, entitled Cartridge For ColorimetricSensor Arrays, with inventor Kenneth S. Suslick, which is incorporatedherein by reference.

Colorimetric arrays that include first and second spots, each includinga different nanoporous pigment, can have increased stability,sensitivity and selectivity toward analytes than conventionalcolorimetric arrays. The nanoporous pigments may be more stable than thecolorants in conventional colorimetric arrays, since the colorants ofthe nanoporous pigments are site-isolated within a nanoporous material,protecting from intermolecular reactions that can occur to solubilizeddyes in solution. Leaching of the colorant into an analyte liquid isalso reduced or eliminated.

Nanoporous pigments may provide increased color intensity for a givenspot in an array, since the loading of the colorant in the spot is notlimited by its solubility. This increased color intensity can providefor improved sensor sensitivity, since the magnitude of the change inspectral properties in response to an analyte can be greater. Nanoporouspigment arrays also may have better sensitivity than conventionalcolorimetric dye arrays. One reason for this increased sensitivity maybe that the nanoporous material acts as pre-concentrator.

The following examples are provided to illustrate one or more preferredembodiments of the invention. Numerous variations may be made to thefollowing examples that lie within the scope of the invention.

EXAMPLES Example 1: Formation of a Nanoporous Pigment Colorimetric ArrayFor Detection, Identification and Quantification of VOCs and TlCs

A colorimetric array for the detection and identification of VolatileOrganic Chemicals (VOCs) and Toxic Industrial Chemicals (TICs) wasmanufactured using a nanoporous material precursor and chemicallyresponsive colorants. The nanoporous material precursor in this examplewas an organically modified silica (ORGAMOSIL) sol-gel solution thatincluded 5 to 50% by volume alkoxysilanes, 5 to 20% by volume water, 1to 80% by volume solvents, 0.001 to 0.1 M hydrochloric acid as acondensation catalyst, and 0.01 to 2% by weight surfactant. The stirredformulation was added to each of the colorants in Table 1, and theresulting mixtures were loaded into a Teflon block containing individualcylindrical wells (⅜″ deep) for each mixture.

A floating slotted dip-pin printer capable of delivering approximately100 nL was used to print the liquids onto the surface of a hydrophobicPVDF membrane. The slotted dip-pin array was dipped into the inkwellwith the formulations filled in the corresponding inkwell holes. The pinarray was then lifted and pressed on a suitable substrate, yielding aprinted array. Once printed, the arrays were cured at room temperaturefor 48 hours.

TABLE 1 Chemoresponsive Colorants Used For Analysis of TICs Spot# Name 12,3,7,8,12,13,17,18-Octaethyl-21H,23H-porphine zinc (II) 25,10,15,20-Tetraphenyl-21H,23H-porphine zinc 3 Zinctetramesitylporphyrin 45,10,15,20-Tetrakis(pentafluorophenyl)-21H,23H-porphine zinc 55,10,15,20-Tetraphenyl-21H,23H-porphine manganese (III) chloride 65,10,15,20-Tetrakis(pentafluorophenyl)-21H, 23H-porphyrin iron (III)chloride 7 5,10,15,20-Tetraphenyl-21H,23H-porphine cobalt (II) 81-[4-[[4-(dimethylamino)phenyl]azo]phenyl]- 2,2,2-trifluoro-ethanone 94-[2-[4-(dimethylamino)phenyl]ethenyl]-2,6- dimethyl-pyryliumperchlorate 10 1-Amino-4-(4-decylphenylazo)-naphthalene 11 Methyl Red +Bu4NOH 12 Phenol Red + Bu4NOH 13 Cresol Red + Bu₄NOH 14 m-CresolPurple + Bu₄NOH 15 Thymol Blue + Bu₄NOH 16 Alizarin + Bu₄NOH 17 BasicFuchsin + Bu₄NOH 18 Crystal Violet 19 Bromocresol Green 20 BromophenolRed 21 Bromothymol Blue 22 Naphthol Blue Black 23 Bromopyrogallol Red 24Pyrocatechol Violet 25 Nile Red 26 Disperse Orange #25 274-(4-Nitrobenzyl)pyridine + N-Benzylaniline 28 Bu₄NBr +Bromochlorophenol Blue 29 ZnOAc2 + m-Cresol Purple + Bu4NOH 30 BasicFuschin + Tosic acid 31 LiNO3 + Cresol Red 32 HgCl2 + Bromophenol Blue +Bu4NOH 33 HgCl₂ + Phenol Red + Bu4NOH 34 Cu(NO3}2 35 AgNO3 + BromocresolGreen 36 AgNO3 + Phenol Red

Example 2: Detection, Identification and Quantification of TICs

The prepared arrays of Example 1 were cut to size and placed inpuncturable, sealed polyacrylic cartridges. Premixed certified gases of16 individual TICs were diluted using digital mass flow controllers totheir immediately dangerous to life or health (IDLH) concentration. Foreach array, the resulting gas stream was contacted with the array in itscartridge. The image of each array was acquired using a flatbed scanner(V200; EPSON, Long Beach, Calif.) before and during exposure to theTICs. Rapid equilibration occurred, and images for analysis wereacquired after two minutes exposure. Upon exposure to the analyte, thearrays underwent reversible reactions that resulted in well definedcolor changes. The RGB values were obtained in a difference map bysubtracting the before image from the after image. To eliminate thepossibility of subtraction artifacts caused by acquisitions near thespot edge, only the spot centers were included in the calculation.Measurements were performed using Photoshop® or ChemEyE™. A database wasassembled from quintuplicate runs of the TICs at IDLH concentrations.Color change profiles, which were unique to each TIC, are shown in thecolor difference maps of FIG. 4.

Different concentrations of TICs were also determined with thecolorimetric array. For purposes of illustration, three TICs (ammonia,sulfur dioxide, and chlorine) were chosen at concentrationscorresponding to their respective IDLH, permissible exposure level(PEL), and sub-PEL concentrations, as listed in FIG. 5. Clearlyidentifiable differences in the color difference maps of FIG. 5 wereobvious, even without statistical analysis.

For statistical analyses [33] of the changes in spectral properties,principal component analysis (PCA) and hierarchical clustering analysis(HCA) were used to analyze the color change profile database. PCAprovides a quantitative evaluation of the analytical dispersion of atechnique based on its number of independent dimensions of variance.Conventional electronic tongue sensors have shown only limitedselectivity, which is believed to be due their relatively low number ofindependent dimensions. Typically, only two dimensions will account formore than 95% of total discrimination in these conventional sensors. Incontrast, there was an extremely high level of dispersion with thecolorimetric arrays of Example 1. When PCA was applied even to thisfamily of very closely related analytes, there were 10 dimensionsnecessary for 90% of total discrimination, as indicated in the graph ofFIG. 6.

FIG. 7 shows the dendrogram generated from the HCA analysis of the datafor quintuplicate tests of 14 TICs at their IDLH, plus a control.Remarkably, all the TICs were accurately clustered with no errors ormisclassifications out of 75 cases.

Example 3: Colorimetric Array For Detection and Identification ofCarbohydrates

Tetramethylorthosilicate (TMOS), methyltrimethoxysilane (MTMS),methanol, and nano-pure water were combined in the molar ratio of1:1:11:5. The mixture was stirred for 2 hours at room temperature. Thestirred formulation was added to the chemoresponsive colorants listed inTable 2, and the mixtures were loaded into a block containing individualcylindrical wells having a depth of ⅜ inch for each mixture. A floatingslotted dip-pin printer capable of delivering approximately 100 nL wasused to print the liquids onto the surface of a nitrocellulose acetatehydrophilic membrane (MILLIPORE, Cat No. SSWP14250, 3.0 μm). The slotteddip-pin array was dipped into the inkwell with the formulations filledin the corresponding inkwell holes. The pin array was then lifted andpressed on a suitable substrate, yielding a printed array. Once printed,the arrays werecured at room temperature for 24 hours and then at 65° C.for 24 hours.

TABLE 2 Chemoresponsive Colorants used for Analysis of Sugars andSweeteners Spot# Name 1 Bromophenol Blue 2 Tetrabromophenol Blue 33′,3″,5′,5″-tetraiodophenosulfonephthalein 4 Bromochlorophenol Blue 5Bromocresol Green 6 Chlorophenol Red 7 Bromophenol Red 8 BromocresolPurple 9 Bromoxylenol Blue 10 Phenol Red 11 m-Cresol Purple 12 XylenolOrange tetrasodium salt 13 Bromopyrogallol Red 14 Methyl Yellow 15 CongoRed 16 Methyl Orange

Example 4: Detection, Identification and Quantification of Sugars andSweeteners

The colorimetric arrays described in Example 3 were tested against 15different sugars (including both mono- and di-saccharides), artificialsweeteners and sugar alcohols. The analytes were D-(−)-Fructose,D-(+)-Galactose, D-(+)-Glucose, -Lactose, Maltitol, D-Mannitol,D-(+)-Mannose, D-(+)-Melibiose, L-Rhamnose, D-(−)-Ribose, Saccharin,Sorbitol, Sucrose, Xylitol, D-(+)-Xylose. Each analyte was dissolved in1 mM phosphate buffer at pH 7.4, with 5 mM 3-nitrophenylboronic acidadded. The concentration of each analyte except for sucrose was 25 mM,and the concentration of sucrose was 150 mM.

For each analyte, the array was placed in a puncturable cartridge, andthe cartridge was placed atop an Epson V200 flatbed photo scanner. Afirst image was obtained with the array exposed to a blank buffersolution. The buffer solution was removed, and a sugar analyte solutionwas injected. After a 5 minute delay, the array was scanned again withthe flatbed photo scanner. The delay ensured complete equilibration ofthe array, since 90% of the equilibration occurred in less than oneminute. FIG. 8 is a graph of the response time of a colorimetric arrayto the sugars and sweeteners, as represented by the change in Euclideandistance over time.

Using the procedures of Example 2, difference maps were obtained foreach analyte, a database was assembled from quintuplicate runs of thesugar analytes, and statistical analysis was performed. Color changeprofiles, which were unique to each analyte, are shown in the colordifference maps of FIG. 9. FIG. 10 shows the HCA dendrogram generatedfrom the HCA analysis of the data. Remarkably, all the carbohydrateswere accurately identified and identified against one another with noerrors or misclassifications out of 80 cases.

Example 5: D-Glucose Concentration Study

In addition to high discrimination, high sensitivity to carbohydrates isessential for most practical applications. For example, thephysiological range of glucose concentrations is from about 2 mM toabout 50 mM; normal fasting plasma glucose (FPG) is about 5 mM and thethreshold of diabetes is above 7 mM. Also, diabetic glucoseconcentrations 2 hours after an oral glucose tolerance test are above11.1 mM [38].

The limit of detection (LOO) of the colorimetric array of Example 3 wasdetermined by contacting the arrays with samples containingconcentrations of 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, and 200 mM of0-glucose in a blank buffer solution. Concentration profiles wereconstructed by plotting the total Euclidean distance of the array colorchange against the 0-Glucose concentration. The overall response of thearray, as measured by the total Euclidean difference vs. 0-glucoseconcentration is shown in FIG. 11. The lower limit of detection (LOO) ofthe array, defined as 3× signal to noise, was <1 mM.

Example 6: D-Glucose Cycling Experiment

While the colorimetric arrays of Example 3 were inexpensive, disposable,and meant for single use, many of the reactions taking place were, infact, reversible. Therefore, the reusability of the arrays was examinedby exposing arrays to a blank buffer solution using a 20 ml/min flowsystem, obtaining a first image, and then cycling to the same bufferinfused with 25 mM 0-glucose, followed by plain buffer again. Thisprocess was repeated for three complete cycles. Due to dead volume andmixing times in the flow apparatus, full equilibration requiredapproximately 6 minutes. The intrinsic response time of the array in theabsence of the dead volume was less than 30 seconds. Surprisingly goodreusability was observed, as shown in the graph of FIG. 12.

While various embodiments of the invention have been described, it willbe apparent to those of ordinary skill in the art that other embodimentsand implementations are possible within the scope of the invention.Accordingly, the invention is not to be restricted except in light ofthe attached claims and their equivalents.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein.

REFERENCES

-   1. Gardner, J. W.; Bartlett, P. N. Electronic Noses: Principles and    Applications; Oxford University Press: New York, 1999.-   2. Albert, K. J.; Lewis, N. S.; Schauer, C. L.; Sotzing, G. A.;    Stitzel, S. E.; Vaid, T. P.; Walt, D. R. Chem. Rev. 2000, 100, 2595.-   3. Lewis, N. S. Accts. Chem. Res. 2004, 37, 663-672.-   4. Johnson, B. A.; Leon M. J. Comp. Neurol. 2007, 503, 1-34.-   5. Anslyn E. V. J. Org. Chem. 2007, 72, 687-699.-   6. Anand, V.; Kataria, M.; Kukkar, V.; Saharan, V.; Choudhury, P. K.    Drug Discovery Today 2007, 12, 257-265.

7. Toke, K. Biomimetic Sensor Technology; Cambridge University Press:Cambridge, UK, 2000.

-   8. Suslick, K. S.; Bailey, D. P.; Ingison, C. K.; Janzen, M.;    Kosal, M. A.; McNamara 111, W. B.; Rakow, N. A.; Sen, A.; Weaver, J.    J.; Wilson, J. B.; Zhang, C.; Nakagaki, S. Quimica Nova 2007, 30,    677-681.-   9. Suslick, K. S. MRS Bull. 2004, 29, 720-725.-   10. Suslick, K. S.; Rakow, N. A.; Sen, A. Tetrahedron 2004, 60,    11133-11138.-   11. Wang, J.; Luthey-Schulten, Z. A.; Suslick, K. S., Proc. Natl.    Acad. Sci. U.S.A. 2003, 100, 3035-3039.-   12. Rakow, N. A.; Suslick, K. S., Nature 2000, 406, 710-713.-   13. Rakow, N. A.; Sen, A.; Janzen, M. C.; Ponder, J. B.;    Suslick, K. S. Angew. Chem. Int. Ed. 2005, 44, 4528-4532.-   14. Janzen, M. C.; Ponder, J. B.; Bailey, D. P.; Ingison, C. K.;    Suslick, K. S. “Colorimetric Sensor Arrays for Volatile Organic    Compounds” Anal. Chem. 2006, 78, 3591-3600.-   15. Zhang, C.; Suslick, K. S., J. Am. Chem. Soc. 2005, 127,    11548-11549.-   16. Zhang, C.; Bailey, D. P.; Suslick, K. S., J. Agric. FoodChem.    2006, 54, 4925-4931.-   17. Zhang, C.; Suslick, K. S. J. Agric. Food Chem., 2007, 55,    237-242.-   18. Suslick, K. S.; Rakow, N. A. “Colorimetric Artificial Nose    Having an Array of Dyes and Method for Artificial Olfaction” U.S.    Pat. No. 6,368,558; Apr. 9, 2002.-   19. Suslick, K. S.; Rakow, N. A.; Sen, A. “Colorimetric Artificial    Nose Having an Array of Dyes and Method for Artificial Olfaction:    Shape Selective Sensors” U.S. Pat. No. 6,495,102 81; Dec. 17, 2002.-   20. Suslick, K. S.; Rakow, N. A.; Sen, A.; McNamara, W. B. Ill;    Kosal, Margaret E. “Colorimetric artificial nose having an array of    dyes and method for artificial olfaction” U.S. Patent Appl.    20030143112; Jul. 21, 2003.-   21. Suslick, K. S.; Rakow, N. A.; Sen, A. “Siloxy porpyhrins and    metal complexes thereof” U.S. Patent Appl. 20030129085; Jul. 10,    2003.-   22. Suslick, K. S. “Colorimetric artificial nose having an array of    dyes and method for artificial olfaction” U.S. Patent Appl.    20030166298; Sep. 4, 2003.-   23. Suslick, K. S.; Rakow, N. A.; Sen, A.; McNamara, W. B. Ill;    Kasal, Margare E. “Colorimetric Artificial Nose having an Array of    Dyes and Method for Artificial Olfaction” U.S. Pat. No. 7,261,857;    Aug. 28, 2007.-   24. Zaggout, F. R., J. Dispersion Sci. Technol. 2005, 26, 757-761.-   25. Rottman, C.; Ottolenghi, M.; Zusman, R.; Lev, O.; Smith, M.;    Gong, G.; Kagan, M. L.; Avnir, D., Mater. Lett. 1992, 13, 293-298.-   26. Zusman, R.; Rottman, C.; Ottolenghi, M.; Avnir, D., J.    Non-Cryst. Solids 1990, 122, 107-109.-   27. Kowada, Y.; Ozeki, T.; Minami, T., J. Sol-Gel Sci. Technol.    2005, 33, 175-185.-   28. Zaggout, F. R.; EI-Nahhal, I. M.; Qaraman, A. E.-F. A.; Al    Dahoudi, N., Mate. Lett. 2006, 60, 3463-3467.-   29. Villegas, M. A.; Pascual, L., Thin Solid Films 1999, 351,    103-108.-   30. Laitinen, H. A. Chemical Analysis (McGraw-Hill: New York, 1960)-   31. Murphy, C. J. et al. Chem. Commun. 2008, 544-557.-   32. Suslick, K. S. et al. Acc. Chem. Res. 2005, 38, 283-291.-   33. Hasswell, S., Practical Guide To Chemometrics; Dekker: NY, 1992.-   34. Lee, J. W.; Lee, J.-S.; Chang, Y.-T., Angew. Chem., Int. Ed.    Engl. 2006, 45, 6485-6487.-   35. Dowlut, M.; Hall, D. G., J. Am. Chem. Soc. 2006, 128, 4226-4227.-   36. Yan, J.; Springsteen, G.; Deeter, S.; Wang, B., Tetrahedron    2004, 60, 11205-11209.-   37. James, T. D.; Sandanayake Samankumara, K. R. A.; Shinkai, S.,    Angew. Chem., Int. Ed. Engl. 1996, 35, 1911-1922.-   38. Larson, P. R.; Kronenberg, H. M.; Melmed, S.; Po/lonsky, K. S.    (eds.) Williams Textbook of Endocrinology, 10th ed. Saunders:    Philadelphia, 2003.

What is claimed is:
 1. A method of making a colorimetric arraycomprising: depositing a first liquid at a first point on a substrate;depositing a second liquid at a second point on the substrate;converting the first liquid into a first spot on the substrate; andconverting the second liquid into a second spot on the substrate,wherein the first spot comprises a first nanoporous pigment and a firstimmobilized, chemoresponsive colorant and the second spot includes thesecond nanoporous pigment and a second immobilized, chemoresponsivecolorant.
 2. The method of claim 1, wherein the first and secondnanoporous pigments are the same, and the first and second immobilized,chemoresponsive colorants are different.
 3. The method of claim 1,wherein the first and second nanoporous pigments are different, and thefirst and second immobilized, chemoresponsive colorants are the same. 4.The method of claim 1, wherein the first and second nanoporous pigmentsare different, and the first and second immobilized, chemoresponsivecolorants are different.
 5. The method of claim 1, wherein the substratecomprises a polymeric membrane.
 6. The method of claim 5, wherein thepolymeric membrane is selected from the group consisting of celluloseacetate, polysulfone, polypropylene, polyvinylidene difluoride (PVDF)membranes, glass, metal, poly(tetrafluoroethylene) (PTFE) andpoly(ethyleneterephthalate) (PET).
 7. The method of claim 1, wherein thefirst and second liquids further comprise at least one ingredientselected from the group consisting of a nanoporous material precursorfor the first nanoporous pigment, a nanoporous material precursor forthe second nanoporous pigment, a solvent, an oxidant, a reductant, acatalyst, additive and a surfactant, or a combination thereof.
 8. Themethod of claim 7, wherein the first and second liquids are each formedaccording to the following process: combining a precursor for anorganically modified silica, a solvent, a nanoporous material precursorfor the first or second nanoporous pigment and the first or secondchemoresponsive colorant to form a mixture; and hydrolyzing the mixtureto form a sol.
 9. The method of claim 1, wherein the first and secondliquids comprise nanoporous material precursors for the first and secondnanoporous pigments, respectively and first and second immobilizedchemoresponsive colorants, respectively.
 10. The method of claim 9,wherein the nanoporous material precursors for the first and secondnanoporous pigments are selected from the group consisting of a polymer,a prepolymer, a precursor for an organically modified silica and aceramic, or a combination thereof.
 11. The method of claim 9, whereinthe nanoporous material precursors for the first and second nanoporouspigments comprise a precursor material for an organically modifiedsilica that has been at least partially hydrolyzed.
 12. The method ofclaim 9, wherein the steps of converting the first and second liquidsinto the first and second spots on the substrate, respectively,comprises solidifying the nanoporous material precursors for the firstand second nanoporous pigments, respectively.
 13. The method of claim12, wherein solidifying the nanoporous material precursors comprisescondensing the nanoporous material precursors to form nanoporous gels ororganically modified silicas.
 14. The method of claim 12, wherein thesolidifying comprises heating the first and second liquids between 10 Cto 30 C from 24 to 72 hours or curing the substrate at a temperaturebetween 10 C to 30 C from 24 to 72 hours.
 15. A colorimetric sensorarray for determining the identity and concentration of volatile organiccompounds (VOCs) and toxic industrial chemicals (TICs), wherein thecolorimetric sensor array comprises a colorimetric array producedaccording to the method of claim
 1. 16. A colorimetric sensor array fordetermining the identity and concentration of a carbohydrate or sugar,wherein the carbohydrate or sugar consists of one member selected fromthe group consisting of a mono-saccharide, a di-saccharide, anartificial sweetener and a sugar alcohol, or a combination thereof,wherein the colorimetric sensor array comprises a colorimetric arrayproduced according to the method of claim
 1. 17. The colorimetric sensorarray according to claim 16, wherein the sugar alcohol consists of onemember selected from the group consisting of D-fructose, D-galactose,D-glucose, β-lactose, maltitol, D-mannitol, D-mannose, D-melibiose,L-rhamnose, D-ribose, saccharin, sorbitol, sucrose, xylitol andD-xylose, or combinations thereof.
 18. A colorimetric sensor array fordetermining the identity and concentration of D-glucose in thephysiological range of glucose concentrations from about 1 mM to about100 mM, wherein the colorimetric sensor array comprises a colorimetricarray produced according to the method of claim
 1. 19. A colorimetricarray for detecting ammonia or other analytes in exhaled breath, whereinthe colorimetric sensor array comprises a colorimetric array producedaccording to the method of claim 1.